The invention relates to the field of molecular genetics and medicine. In particular the present invention relates to the field of gene therapy, more in particular to gene therapy using adenoviruses.
At present in gene therapy, genetic information is delivered to a host cell in order to either correct (supplement) a genetic deficiency in the cell, or to inhibit an unwanted function in the cell, or to eliminate the host cell. Of course the genetic information can also be intended to provide the host cell with a wanted function, for instance to supply a secreted protein to treat other cells of the host, etc. Thus there are at least three different approaches in gene therapy, one directed towards compensating a deficiency present in a (mammalian) host; the second directed towards the removal or elimination of unwanted substances (organisms or cells); and the third towards providing a cell with a wanted function.
For the purpose of gene therapy, adenoviruses have been proposed as suitable vehicles to deliver genes to the host. Gene-transfer vectors derived from adenoviruses (so-called adenoviral vectors) have a number of features that make them particularly useful for gene transfer. 1) The biology of the adenoviruses is characterized in detail, 2) the adenovirus is not associated with severe human pathology, 3) the virus is extremely efficient in introducing its DNA into the host cell, 4) the virus can infect a wide variety of c ells and has a broad host-range, 5) the virus can be produced at high virus titers in large quantities, and 6) the virus can be rendered replication defective by deletion of the early-region 1 (E1) of the viral genome (Brady and Crystal 1994).
However, there are still drawbacks associated with the use of adenoviral vectors especially the well investigated serotypes of subgroup C adenoviruses. These serotypes require the presence of the Coxsackie adenovirus receptor (CAR) on cells for successful infection. Although this protein is expressed by many cells and established cell lines, this protein is absent on many other primary cells and cell lines making the latter cells difficult to infect with serotypes 1, 2, 5, and 6.
The adenovirus genome is a linear double-stranded DNA molecule of approximately 36000 base pairs. The adenovirus DNA contains identical Inverted Terminal Repeats (ITR) of approximately 90-140 base pairs with the exact length depending on the serotype. The viral origins of replication are within the ITRs exactly at the genome ends. Most adenoviral vectors currently used in gene therapy have a deletion in the E1 region, where novel genetic information can be introduced. The E1 deletion renders the recombinant virus replication defective. It has been demonstrated extensively that recombinant adenovirus, in particular serotype 5 is suitable for efficient transfer of genes in vivo to the liver, the airway epithelium and solid tumors in animal models and human xenografts in immunodeficient mice (Bout 1996; Blaese et al. 1995). At present, six different subgroups of human adenoviruses have been proposed which in total encompasses 51 distinct adenovirus serotypes. Besides these human adenoviruses an extensive number of animal adenoviruses have been identified (Ishibashi and Yasue 1984).
A serotype is defined on the basis of its immunological distinctiveness as determined by quantitative neutralization with animal antisera (horse, rabbit). If neutralization shows a certain degree of cross-reaction between two viruses, distinctiveness of serotype is assumed if A) the hemagglutinins are unrelated, as shown by lack of cross-reaction on hemagglutination-inhibition, or B) substantial biophysical/biochemical differences in DNA exist (Francki et al. 1991). The nine serotypes identified last (42-51) were isolated for the first time from HIV-infected patients (Hierholzer et al. 1988; Schnurr and Dondero 1993; De Jong et al. 1999). For reasons not well understood, most of such immuno-compromised patients shed adenoviruses that were rarely or never isolated from immuno-competent individuals (Hierholzer et al. 1988; Hierholzer 1992; Khoo et al. 1995, De Jong et al. 1999).
At present the adenovirus serotype 5 is most widely used for gene therapy purposes. Similar to serotypes 2, 4 and 7, serotype 5 has a natural affiliation towards lung epithelia and other respiratory tissues. In contrast, it is known that, for instance, serotypes 40 and 41 have a natural affiliation towards the gastrointestinal tract. For a detailed overview of the disease association of the different adenovirus serotype see Table I. In this Table I there is one deviation from the literature. Sequence analysis and hemagglutination assays using erythrocytes from different species performed in our institute indicated that in contrast to the literature (De Jong et al. 1999) adenovirus 50 proved to be a D group vector whereas adenovirus 51 proved to be a B-group vector.
The natural affiliation of a given serotype towards a specific organ can either be due to a difference in the route of infection i.e., make use of different receptor molecules or internalization pathways. However, it can also be due to the fact that a serotype can infect many tissues/organs but it can only replicate in one organ because of the requirement of certain cellular factors for replication and hence clinical disease. At present it is unknown which of the above mentioned mechanisms is responsible for the observed differences in human disease association. However it is known that different adenovirus serotypes can bind to different receptors due to sequence dissimilarity of the capsid proteins, e.g. fiber proteins. For instance, it has been shown that adenoviruses of subgroup C such as Ad2, and Ad5 bind to different receptors as compared to adenoviruses from subgroup B such as Ad3 (Defer et al. 1990). An adenovirus from subgroup B is referred to as a B-type adenovirus. Likewise, it was demonstrated that receptor specificity could be altered by exchanging the Ad3 with the Ad 5 knob protein, and vice versa (Krasnykh et al. 1996; Stevenson et al. 1995 and 1997). The C-terminus of the fiber protein, or knob, is responsible for initial interaction with the cellular adenovirus receptor. Thus the fiber protein is mainly responsible for receptor specificity. As different host cells can have different receptors, the fiber protein largely determines at which host cells the adenovirus preferably binds. The preference for binding to a certain kind of host cell is called a tropism. If an adenovirus has a tropism for a certain host cell, it may, or may not, bind to other kind of cells as well. The tropism of an adenovirus is thus at least partly dependent on the kind of fiber protein, and/or knob protein.
In the United States alone 95,000 knee replacements and 41,000 other surgical procedures to repair cartilaginous defects of the knee are performed on an annual basis. This, together with other cartilage diseases (i.e. joint surface irregularities, craniofacial deformation, osteogenesis imperfecta, meniscal injury, anencephaly, intra articular fractures, osteoporosis, osteoarthritis, spinal cord fusion, and rheumatoid arthritis) warrant the enormous interest in understanding the underlying biological and biochemical defects of the diseases as well as the interest in gene therapy as a possible cure (reviewed in Frenkel and DiCesare 1999). The strategies to treat these diseases are diverse ranging from direct delivery of genes to sites of injuries, to cell-based delivery approaches, or ex vivo tissue engineering. In case genes are directly delivered to a site of injury either retroviruses, adenoviruses, naked DNA, or liposome complexed DNA are contemplated (Madry and Trippel 2000; Lubberts et al. 1999; Goto et al 1999). The DNA can encode either for amino acid sequences that inhibit the disease progression and/or amino acid sequences that counteract the loss of cartilage. Non-limiting examples of genes that inhibit disease progression are TGFbeta (Nishida et al. 1999), IL-4 (Lubberts et al. 1999), p16INK4a (Taniguchi et al. 1999), IL-I (Fernandez et al. 1999), IL-10 (Whalen et al. 1999), or substances (anti-inflammatory drugs, TNFa, immunosuppressive agents) which can down regulate the activity of NOS or COX (Amin et al. 1999). Both other strategies, cell based or ex vivo bioengineering, use the same genes (Gazit et al. 1999): two pleiotropic inflammatory mediators overproduced in arthritis infected joints. Non-limiting examples of genes that counteract the cartilage degradation are the family of bone morphogenesis proteins (Mason et al. 1998; Kramer et al 2000, Pizette and Niswander 2000). Cells of choice to perform cell based delivery at sites of injury or transplanted into a scaffold are chondrocytes or mesenchymal stem cells derived from human bone marrow (Richardson et al. 1999, Silverman et al. 2000, Gazit et al. 1999). For all these strategies to become therapeutically interesting the delivery of gene sequences chondrocytes needs to be very efficient such that a) expression levels of the therapeutic genes are high and b) low dosages of the gene transfer vehicle, i.e. adenoviruses can be applied to circumvent possible vector-mediated toxic effects.